The development of antimicrobial agents led to a significant decrease in morbidity and mortality from infectious diseases in this century. This accomplishment was largely due to the widespread use of the major classes of antibiotics, such as the sulfonamides, penicillins, cephalosporins, aminoglycosides, and tetracyclines (Goodman et al., “The Pharmacological Basis of Therapeutics”, Macmillan Publishing, New York, 1985). However, in recent years, the trend in reducing infectious disease mortality has been threatened by the emergence of resistant strains of microorganisms that are no longer susceptible to the currently available antimicrobial agents. As a result, maintenance of public health requires that new antimicrobial agents be developed to counter these emerging resistant strains in order to prevent the diseases that have previously considered to be under control from reemerging.
Biologically active peptides, such as antimicrobial peptides (hereinafter “AMPs), have little chance to develop resistance because the antimicrobial peptides show activity by a mechanism that is totally different from that of the conventional antibiotics. The AMPs are the biophylaxis systems of the species which act to defend or protect itself from the infection by virus or bacteria.
AMPs are low molecular weight natural peptides that exhibit antimicrobial activity. They are part of the innate immune response of plants, invertebrates and vertebrates. Different AMPs have been reported to be isolated from natural sources since 1930s. For example, in 1939 Dubos demonstrated that a soil bacillus, subsequently identified as B. brevis, produced substances that could prevent pneumococcal infections in mice. Subsequently, Hotchkiss and Dubos purified two substances composed of amino acids and one of these, gramicidin, became available as a therapeutic agent. Subsequent studies on antimicrobial peptides have identified many active agents (Moberg and Cohn (eds), (1990), “Launching the Antibiotic Era. Personal accounts of the discovery and use of the first antibiotics.” Rockefeller University Press, New York).
The variety and diversity of the AMPs have been expanding. Cationic peptides are the most widespread form of AMPs, while anionic peptides, aromatic dipeptides, processed forms of oxygen-binding proteins, and processed forms of natural structural and functional proteins have all been reported. In spite of the astonishing diversity in structure and chemical nature among the AMPs, they all present antimicrobial activity. AMPs participate in host defense reactions against invading microorganisms such as bacteria, fungi, parasites, and enveloped virus.
To date, the AMPs are temporarily grouped into four distinct families based on biochemical characteristics:
(I) Linear cationic basic peptides forming amphipathic α-helices, such as the cecropins, the first antimicrobial peptide isolated from insect hemolymph (Boman and Hultmark, (1987), Ann. Rev. Microbiol. 41:103-126); and the magainins found in the skin of Xenopus laevis (Zasloff, (1987), Proc. Natl. Acad. Sci. USA, 84:5449-5453).
(II) Peptides with one to six intramolecular disulfide linkages, such as the defensins (Hoffmann and Hetru, (1992), Immunol. Today 13:411-415); antifungal peptides from Drosophila, drosomycin (Fehlbaum et al., (1994), J. Biol. Chem. 269:33159-33163); thanatin from Podisus (Fehlbaum et al., (1996), Proc. Natl. Acad. Sci., USA, 93:1221-1225); tachyplesin, big defensin, and tachycitin from limulus (Nakamura et al., (1988), J. Biol. Chem. 263:16709-16713); and other cysteine-rich antimicrobial peptides isolated from a scorpion (Ehret-Sabatier et al., (1996), J. Biol. Chem. 271:29537-29544) and a bivalve mollusk (Charlet et al., (1996), J. Biol. Chem. 272:28398-28406).
(III) Proline-rich peptides, such as apidaecins and abaecins from Hymenoptera (Casteels et al., (1990), Eur. J. Biochem. 187:381-386); and drosocin from Drosophila hemolymph (Bulet et al., (1993), J. Biol. Chem. 268:14893-14897).
(IV) Glycine-rich antimicrobial peptides or polypeptides (9-30 kDa), such as the attacins (Hultmark, et al., (1983), EMBO J. 2:571-576), diptericin (Dimarcq et al., (1988), Eur. J. Biochem. 171:17-22), and sarcotoxins (Kanai and Natori, (1990), Mol. Cell. Biol. 10(12): 6114-22).
Although AMPs have been discovered in a variety of animals and plants, very few findings of AMPs in the Crustaceans have been reported, probably due primarily to their unique immune system. Among the Crustaceans, penaeid shrimp represents one of the fastest growing Crustaceans in the world. Penaeid shrimp belong to the largest phylum in the animal kingdom, the Arthropoda. This group of animals is characterized by the presence of paired appendages and a protective cuticle or exoskeleton that covers the whole animal. The subphylum Crustacea is made up of 42,000, predominantly aquatic, species, that belong to 10 classes. Within the class Malacostraca, shrimp, together with crayfish, lobsters and crabs, belong to the order Decapoda.
Tiger shrimp (Penaeus monodon Fabricius), also known as black tiger shrimp, black tiger prawn, giant tiger shrimp, is one of the most important cultivated shrimp species in the marine shrimp aquaculture industry around the world. Tiger shrimp belongs to the Family of Penaeidae Rafinesque, Genus of Penaeus Fabricius, Subgenus of Penaeus, and Species monodon. Other important cultured penaeid shrimp species include Pacific white shrimp (P. vannamei), kuruma shrimp (P. japonicus), blue shrimp (P. stylirostris), and Chinese white shrimp (P. chinensis). World shrimp production is dominated by P. monodon, which accounted for more than 50% of the production in 1999. Tiger shrimp is widely distributed throughout the greater part of the Indo-Pacific region, ranging northward to Japan and Taiwan, eastward to Tahiti, southward to Australia, and westward to Africa.
The giant black tiger shrimp (P. monodon) derived its name from the huge size and banded tail, providing a tiger-striped appearance to this species. It is by far the largest, reaching 330 mm or more in body length, and exhibits the highest growth rate, of all cultured penaeids. (Lee and Wickins, (1992), Blackwell Scientific Publications; The University press, Cambridge, 392 pp). P. monodon can reach a market size up to 25-30 g within 3-4 months after postlarvae stocking in culture ponds and tolerates a wide range of salinities. Although P. monodon was normally considered as exceptionally tough, the rapid growth and intensification of its culture industry generated crowding and increased environmental degradation, which made the animals more susceptible for diseases.
The shrimp aquaculture industry has suffered huge economic loss due to diseases, which mainly caused by viruses and bacteria, and to a lesser extent, rickettsiae, fungi, and parasites. For example, the white spot syndrome virus (WSSV) has had a great impact on shrimp culture and at present still causes major problem in P. monodon. Other important viruses include infectious hypodermal and haematopoietic necrosis (IHHN) virus, hepatopancreatic parvovirus (HPV), baculoviral midgut gland necrosis (BMN) virus, baculovirus penaei (BP), yellow head virus (YHV), monodon baculovirus (MBV), lymphoid organ vacuolisation virus (LOVV) and Taura syndrome virus. Viral diseases are often accompanied by bacterial infestations. However, only a small number of bacterial species have been diagnosed as infectious agents in penaeid shrimp. Vibrio spp. are by far the major bacterial pathogens and can cause severe mortality, particularly in hatcheries. Vibriosis is often considered to be a secondary (opportunistic) infection, which usually occurs when shrimp are weakened. Primary pathogens can kill even when other environmental factors are adequate, whereas opportunistic pathogens are normally present in the natural environment of the host and only kill when other physiological or environmental factors are poor. However, the differences in effects between primary pathogens, such as the WSSV, and secondary pathogens, such as Vibrio spp., are marginal, primarily due to lack of basic knowledge of the interaction between the pathogens of cultivated shrimp and the reaction of the hosts. In fact, the transmission of diseases in an intensive shrimp culture environment is extremely easy, because of the dense culture conditions. Thus, losses of cultivated penaeid shrimp due to diseases, whether slow continuous attrition or sudden catastrophic epizootics, are familiar problems that confront the aquaculture sector.
So far, only a few researches on AMPs have been performed in crustacean. For example, penaeidins, a family of AMPs from the white shrimp (Penaeid vannamei), have been isolated. (Destoumieux et al., (2000), CMLS, Cell. Mol. Life. Sci. 57:1260-1271). However, the AMPs of the tiger shrimp (Penaeid Monodon) remain largely unknown.
In the invention to be presented in the following sections, a novel AMP isolated from P. monodon, monodoncin, is described. This AMP has a wide-range of antimicrobial activity, which is particularly important to diseases control to ensure the long term survival of not only penaeid shrimp, but also other aquatic species as well as humans and domestic animals, such as chicken and swine. The investigation of this AMP further provides insights with regard to the understanding of the innate immune system of crustaceans.